Polypeptide Containing DNA-Binding Domain

ABSTRACT

The present invention provides an artificial nuclease comprising a DNA-binding domain and a function domain linked to each other via a polypeptide consisting of 35 to 55 amino acid residues wherein amino acid residues at two sites in a DNA-binding module contained in a DNA-binding domain exhibit a mode of repetition that is different for every four DNA-binding modules; a vector for expressing said artificial nuclease; a vector library for preparing said vector; and a vector set for preparing said vector library.

A computer readable text file, entitled “SequenceListing.txt,” createdon or about Jan. 28, 2016 with a file size of about 46 kb contains thesequence listing for this application and is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention relates to a polypeptide containing a DNA-bindingdomain and a function domain. The present invention also relates to avector comprising a polynucleotide coding for said polypeptide. Thepresent invention further relates to a vector library for preparing saidvector. The present invention also relates to a vector set for preparingsaid vector library. The present application claims the priority basedon Japanese patent application No. 2013-166768 filed on Aug. 9, 2013, awhole content of which is incorporated herein by reference.

BACKGROUND ART

For a polypeptide comprising a plurality of nuclease subunits consistingof a DNA-binding domain and a function domain, TALEN (TALE Nuclease),ZFN (Zinc Finger Nuclease) and the like are known (Patent references1-4, Non-patent references 1-5). For instance, an artificial nuclease isknown in which a DNA-cleaving domain is used as a function domain. Theseartificial nucleases cause cleavage of DNA duplicates by a multimerformed by a plurality of DNA-cleaving domains approaching close togetheraround a binding site of a DNA-binding domain. A DNA-binding domaincomprises repetition of plurality of DNA-binding modules and therespective DNA-binding module recognizes a specific base pair in a DNAstrand. Thus, by suitably designing DNA-binding modules, it becomespossible to specifically cleave a sequence of interest. By utilizingerrors and recombinations that occur when sequence specific cleavage issubject to repair, it is possible to introduce deletion, insertion andmutation of a gene on a genomic DNA. Therefore, these nucleases may beapplied for various genetic modifications such as a genome editing (cf.Non-patent reference 6).

Non-patent reference 1 discloses a polypeptide in which a DNA-bindingdomain and a function domain are linked to each other via a linker of 47amino acid residues. Non-patent reference 1 discloses a nuclease inwhich a single amino acid residue in a specific site in a DNA-bindingmodule other than a DNA recognition site is periodically varied for theevery four DNA-binding modules used. However, Non-patent reference 1fails to disclose that the used polypeptide has compatibility between ahigh level of desired function of a function domain and a high level ofspecificity of sequence recognition of a DNA binding domain. Non-patentreference 1 also fails to disclose that a plurality of amino acidresides in a DNA-binding module are periodically varied. Non-patentreference 1 also fails to disclose the significance and the purpose ofthe periodical variation.

Non-patent reference 2 discloses a polypeptide in which a DNA-bindingdomain and a function domain are linked to each other via a linker of 63amino acid residues. Non-patent reference 2 also discloses a polypeptidein which amino acid residue(s) in a DNA-binding module other than thoseamino acid residues in a DNA recognition site is/are periodically variedfor the every four DNA-binding modules used. However, Non-patentreference 2 fails to disclose that the used polypeptide hascompatibility between a high level of desired function of a functiondomain and a high level of specificity of sequence recognition of aDNA-binding domain. Non-patent reference 2 also does not refer toanything about the purpose and the significance of the periodicalvariation for every four DNA-binding modules.

Non-patent reference 3 discloses a polypeptide in which a DNA-bindingdomain and a function domain are linked to each other via a linker of 47amino acid residues. Non-patent reference 3 also discloses a polypeptidein which amino acid residue(s) in a DNA-binding module other than thoseamino acid residues in a DNA recognition site is/are varied at randomfor the every DNA-binding modules used. However, Non-patent reference 3fails to disclose that the used polypeptide has compatibility between ahigh level of desired function of a function domain and a high level ofspecificity of sequence recognition of a DNA-binding domain. Non-patentreference 3 also fails to disclose the purpose and the significance ofthe variation at random. Non-patent reference 3 also fails to disclosethe periodical variation for every DNA-binding module.

Non-patent reference 4 discloses a polypeptide in which a DNA-bindingdomain and a function domain are linked to each other via a linker of 63amino acid residues. Non-patent reference 5 discloses a polypeptide inwhich a DNA-binding domain and a function domain are linked to eachother via a linker of 47 amino acid residues. However, neitherNon-patent reference 4 nor Non-patent reference 5 discloses that theused polypeptide has compatibility between a high level of desiredfunction of a function domain and a high level of specificity ofsequence recognition of a DNA-binding domain. Also, neither Non-patentreference 4 nor Non-patent reference 5 discloses the periodicalvariation for every DNA-binding module.

PATENT REFERENCES

-   Patent reference 1: WO 2011/072246-   Patent reference 2: WO 2011/154393-   Patent reference 3: WO 2011/159369-   Patent reference 4: WO 2012/093833

NON-PATENT REFERENCES

-   Non-patent reference 1: Nucleic Acids Res. 2011 November;    39(21):9283-93.-   Non-patent reference 2: Nat Biotechnol. 2011 Aug. 5; 29(8):697-8.-   Non-patent reference 3: Nat Biotechnol. 2011 February; 29(2):143-8.-   Non-patent reference 4: Nature. 2012 Nov. 1; 491(7422):114-8.-   Non-patent reference 5: Genes Cells. 2013 April; 18(4):315-26.-   Non-patent reference 6: Cell. 2011 Jul. 22; 146(2):318-31.

DISCLOSURE OF THE INVENTION Technical Problem to be Solved by theInvention

It is expected that efficiency for obtaining a desired result isimproved when a polypeptide comprising a DNA-binding domain with highfunction of a function domain is used. For instance, in case of anartificial nuclease comprising a DNA-binding domain and a DNA-cleavingdomain, it is expected that probability of DNA cleavage is improved toimprove efficiency for obtaining cells with a genetic modification ofinterest when such a nuclease as having a high DNA cleavage activity isused. However, a conventional polypeptide comprising a DNA-bindingdomain, when showing a high activity of a function domain, is likely toexert a high function also in the region other than a target nucleotidesequence of a DNA-binding domain and thus is not appropriate in view ofsafety. As such, it was difficult to establish compatibility between ahigh level of specificity of DNA sequence recognition and a high levelof function of a function domain. Besides, cumbersome procedures such asintroduction of repetition of DNA-binding modules corresponding totarget sequences into a vector are necessary for preparing a polypeptidecomprising a DNA-binding domain. Thus, there is a need for a polypeptidethat can be prepared with simpler procedures more rapidly.

Therefore, an object of the present invention is to provide apolypeptide, which has compatibility between a high level of function ofa function domain and a high level of specificity of DNA sequencerecognition, can safely exert desired function with high probability,and can be prepared with simple procedures.

Means for Solving the Problems

The present inventors have earnestly studied to solve the above problemsand as a result have found that a polypeptide wherein a DNA-bindingdomain and a function domain are linked to each other via a polypeptideconsisting of 35 to 55 amino acid residues and wherein amino acidresidues at two specific sites in a DNA-binding module contained in aDNA-binding domain exhibit a mode of repetition that is different forevery four DNA-binding modules has compatibility between a high level offunction of a function domain and a high level of specificity of DNAsequence recognition. A vector for expressing said polypeptide could beprepared with simple procedures by using a vector set of specificfeatures and a vector library of specific features.

Thus, in the first aspect, the present invention provides a polypeptidecomprising a DNA-binding domain and a function domain,

wherein the DNA-binding domain and the function domain are linked toeach other via a polypeptide consisting of 35 to 55 amino acid residues,

wherein the DNA-binding domain comprises a plurality of DNA-bindingmodules consecutively from the N-terminus,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position4n−3 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position4n−2 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position4n−1 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 4ncounted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position4n−3 counted from the N-terminus, a combination of an amino acid residueat position x and an amino acid residue at position y in a DNA-bindingmodule at position 4n−2 counted from the N-terminus, a combination of anamino acid residue at position x and an amino acid residue at position yin a DNA-binding module at position 4n−1 counted from the N-terminus,and a combination of an amino acid residue at position x and an aminoacid residue at position y in a DNA-binding module at position 4ncounted from the N-terminus are different from each other, and

wherein n is natural number of 1 to 10, x is natural number of 1 to 40,y is natural number of 1 to 40, and x and y are different natural numberfrom each other.

In the second aspect, the present invention provides the polypeptide ofthe first aspect wherein the function domain is a DNA-cleaving domain.

In the third aspect, the present invention provides a vector comprisinga polynucleotide coding for the polypeptide of the first aspect or thesecond aspect.

In the fourth aspect, the present invention provides a vector libraryfor preparing the vector of the third aspect,

wherein the vector library consists of a plurality of vectors, each ofwhich vector has a first restriction site, a polynucleotide coding forfour DNA-binding modules and a second restriction site in this orderfrom the 5′-end,

wherein a combination of the first restriction site and the secondrestriction site is any one of a combination of a restriction site oftype A and a restriction site of type B, a combination of a restrictionsite of type A and a restriction site of type C, a combination of arestriction site of type A and a restriction site of type D, acombination of a restriction site of type A and a restriction site oftype E, a combination of a restriction site of type B and a restrictionsite of type C, a combination of a restriction site of type C and arestriction site of type D, and a combination of a restriction site oftype D and a restriction site of type E,

wherein each of the restriction sites of types A to E produce differentcleaved terminals when cleaved with the same restriction enzyme; andamong the four DNA-binding modules,

wherein, a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 1counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 2counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 3counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 4counted from the 5′-terminus is the same for any of the vectors, and

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 1counted from the 5′-terminus, a combination of an amino acid residue atposition x and an amino acid residue at position y in a DNA-bindingmodule at position 2 counted from the 5′-terminus, a combination of anamino acid residue at position x and an amino acid residue at position yin a DNA-binding module at position 3 counted from the 5′-terminus, anda combination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4 counted fromthe 5′-terminus are different from each other, and

wherein x is natural number of 1 to 40, y is natural number of 1 to 40,and x and y are different natural number from each other.

Furthermore, in the fifth aspect, the present invention provides avector set for preparing the vector library of the fourth aspect,

wherein the vector set comprises a plurality of vectors, each of whichvector comprises a first restriction site, a DNA-binding module and asecond restriction site in this order from the 5′-end,

wherein the first restriction site and the second restriction siteproduce different cleaved terminals when cleaved with the samerestriction enzyme,

wherein a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module is any of thefour different combinations, and

wherein x is natural number of 1 to 40, y is natural number of 1 to 40,and x and y are different natural number from each other.

In the sixth aspect, the present invention provides a vector comprisinga polynucleotide coding for a polypeptide comprising a DNA-bindingdomain and a function domain,

wherein the DNA-binding domain and the function domain are linked toeach other via a polypeptide consisting of 40 to 50 amino acid residues,

wherein the DNA-binding domain comprises 16 to 20 DNA-binding modulesconsisting of 34 amino acid residues consecutively from the N-terminus,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position4n−3 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position4n−2 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position4n−1 counted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 4ncounted from the N-terminus is the same for any of n,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position4n−3 counted from the N-terminus, a combination of an amino acid residueat position 4 and an amino acid residue at position 32 in a DNA-bindingmodule at position 4n−2 counted from the N-terminus, a combination of anamino acid residue at position 4 and an amino acid residue at position32 in a DNA-binding module at position 4n−1 counted from the N-terminus,and a combination of an amino acid residue at position 4 and an aminoacid residue at position 32 in a DNA-binding module at position 4ncounted from the N-terminus are different from each other,

wherein n is natural number of 1 to 5, and

wherein the DNA-binding domain is from TALE.

In the vector, the function domain is preferably a DNA-cleaving domain.

In the seventh aspect, the present invention provides a vector libraryfor preparing the vector as set forth in the sixth aspect above,

wherein the vector library consists of a plurality of vectors, each ofwhich vector has a first restriction site, a polynucleotide coding forfour DNA-binding modules and a second restriction site in this orderfrom the 5′-end,

wherein a combination of the first restriction site and the secondrestriction site is any one of a combination of a restriction site oftype A and a restriction site of type B, a combination of a restrictionsite of type A and a restriction site of type C, a combination of arestriction site of type A and a restriction site of type D, acombination of a restriction site of type A and a restriction site oftype E, a combination of a restriction site of type B and a restrictionsite of type C, a combination of a restriction site of type C and arestriction site of type D, and a combination of a restriction site oftype D and a restriction site of type E,

wherein each of the restriction sites of types A to E produce differentcleaved terminals when cleaved with the same restriction enzyme; andamong the four DNA-binding modules,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 1counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 2counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 3counted from the 5′-terminus is the same for any of the vectors,

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 4counted from the 5′-terminus is the same for any of the vectors, and

wherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a DNA-binding module at position 1counted from the 5′-terminus, a combination of an amino acid residue atposition 4 and an amino acid residue at position 32 in a DNA-bindingmodule at position 2 counted from the 5′-terminus, a combination of anamino acid residue at position 4 and an amino acid residue at position32 in a DNA-binding module at position 3 counted from the 5′-terminus,and a combination of an amino acid residue at position 4 and an aminoacid residue at position 32 in a DNA-binding module at position 4counted from the 5′-terminus are different from each other.

In the eighth aspect, the present invention provides a vector set forpreparing the vector library as set forth in the seventh aspect above,

wherein the vector set comprises a plurality of vectors, each of whichvector comprises a first restriction site, a DNA-binding module and asecond restriction site in this order from the 5′-end,

wherein the first restriction site and the second restriction siteproduce different cleaved terminals when cleaved with the samerestriction enzyme,

wherein the first restriction site and the second restriction site arethe ones not cleaved by a restriction enzyme that cleaves the firstrestriction site and the second restriction site contained in thevectors constituting the vector library as set forth in claim 3, andwherein a combination of an amino acid residue at position 4 and anamino acid residue at position 32 in a

DNA-binding module is any of the four different combinations.

In the ninth aspect, the present invention provides a method forpreparing a modified cell which comprises introducing the vector as setforth in the sixth aspect above into a cell followed by expression.

In the tenth aspect, the present invention provides a method forpreparing a modified cell which comprises introducing the vector as setforth in the seventh aspect above into a cell followed by expression.

In the eleventh aspect, the present invention provides a modified cellproduced by the method as set forth in the ninth aspect above.

In the twelfth aspect, the present invention provides a cell withmutation on the genome being introduced produced by the method as setforth in the tenth aspect above.

In the thirteenth aspect, the present invention provides a cell withmodification by the vector as set forth in the sixth aspect above.

In the fourteenth aspect, the present invention provides a cell withmutation on the genome by the vector as set forth in the seventh aspectabove.

In the fifteenth aspect, the present invention provides a plant or ananimal comprising the cell as set forth in any one of the eleventh tofourteenth aspects above.

Effects of the Invention

The polypeptide of the present invention accomplishes a high level offunction of a function domain and simultaneously a high level ofspecificity of DNA sequence recognition. Thus, by introducing a vectorcomprising a polynucleotide coding for the polypeptide of the presentinvention into cells, a desired result can be attained safely with highprobability. Besides, by using the vector library of the presentinvention, a vector for expressing a polypeptide that has compatibilitybetween a high level of function of a function domain and a high levelof specificity of DNA sequence recognition can be prepared with simpleprocedures rapidly. Furthermore, by using the vector set of the presentinvention, the vector library of the present invention can be preparedwith simple procedures.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an illustration showing structural features and process forthe preparation of the vector set, the vector library and the vector ofthe present invention.

FIG. 2 shows an embodiment of an amino acid sequence and a nucleotidesequence of a DNA-binding module contained in the vector set, the vectorlibrary and the vector of the present invention.

FIG. 3 shows an embodiment of an amino acid sequence and a nucleotidesequence contained in the vector of the present invention.

FIG. 4A shows structural features of the vectors prepared by the methodsof Example 3, and Comparative Examples 1 to 3.

FIG. 4B shows structural features of the vectors prepared by the methodsof Example 3, and Comparative Examples 1 to 3.

FIG. 4C shows comparison of sequence specificity of nucleases expressedby a combination of various vectors.

FIGS. 5A and 5B show a design of vectors prepared by the methods ofExample 3, and Comparative Examples 1 to 3 (FIG. 5A) and comparison of alevel of a DNA cleavage activity of nucleases expressed by these vectors(FIG. 5B).

FIGS. 6A and 6B show a design of vectors prepared by the methods ofExample 3, and Comparative Examples 1 to (FIG. 6A) and comparison ofsequence specificity of nucleases expressed by these vectors (FIG. 6B).

BEST MODE FOR CARRYING OUT THE INVENTION

In the first aspect, the present invention provides a polypeptidecomprising a DNA-binding domain and a function domain. A DNA-bindingdomain may be derived from TALE (Transcription Activator-Like Effector)of plant pathogen Xanthomonas, Zinc finger and the like.

A function domain includes a domain coding for an enzyme, atranscriptional regulatory element, a reporter protein and the like. Theenzyme includes a DNA modification enzyme such as recombinase, nuclease,ligase, kinase, phosphatase; and other enzymes such as lactamase and thelike. As used herein, a domain coding for nuclease is referred to as aDNA-cleaving domain. The transcriptional regulatory element includesactivator, repressor and the like. The reporter protein includes afluorescent protein such as green fluorescent protein (GFP), humanizedRenilla green fluorescent protein (hrGFP), enhanced green fluorescentprotein (eGFP), enhanced blue fluorescent protein (eBFP), enhanced cyanfluorescent protein (eCFP), enhanced yellow fluorescent protein (eYFP),red fluorescent protein (RFP or DsRed), mCherry and the like; abioluminescent protein such as firefly luciferase, Renilla luciferaseand the like; an enzyme converting a chemiluminescent substrate such asalkaline phosphatase, peroxidase, chloramphenicol acetyltransferase,β-galactosidase and the like. A DNA-cleaving domain is preferably theone that is close by another DNA-cleaving domain to form a multimer toobtain an improved nuclease activity. Such a DNA-cleaving domainincludes those from FokI and the like.

In the first aspect of the polypeptide of the present invention, aDNA-binding domain and a function domain are linked to each other via apolypeptide consisting of 35-55, preferably 40-50, more preferably45-49, most preferably 47 amino acid residues. A polypeptide throughwhich a DNA-binding domain and a function domain are linked to eachother includes, for instance, a polypeptide consisting of the amino acidsequence of from position 754 to position 801 of SEQ ID NO: 34 as wellas a polypeptide having sequence identity of 85%, 90%, 95%, or 97% withthe amino acid sequence of from position 754 to position 801 of SEQ IDNO: 34.

In the first aspect of the polypeptide of the present invention, aDNA-binding domain comprises a plurality of DNA-binding modulesconsecutively from the N-terminus. A single DNA-binding modulerecognizes specifically a single base pair. The number of a DNA-bindingmodule contained in a DNA-binding domain is preferably 8-40, morepreferably 12-25, even more preferably 15-20 in view of compatibilitybetween a high level of function of a function domain and a high levelof specificity of DNA sequence recognition. A DNA-binding moduleincludes, for instance, TAL effector repeat and the like. The length ofa DNA-binding module includes, for instance, 20-45, 30-38, 32-36, or 34.The length of DNA-binding modules contained in a DNA-binding domain ispreferably the same for all the DNA-binding modules. A DNA-bindingmodule includes, for instance, a polypeptide consisting of the aminoacid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. When the aminoacid residues at positions 12 and 13 in SEQ ID NOs:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, or 32 are H and D, respectively,said DNA-binding domain recognizes C as a nucleotide. When the aminoacid residues at positions 12 and 13 are N and G, respectively, saidDNA-binding domain recognizes T as a nucleotide. When the amino acidresidues at positions 12 and 13 are N and I, respectively, saidDNA-binding domain recognizes A as a nucleotide. When the amino acidresidues at positions 12 and 13 are N and N, respectively, saidDNA-binding domain recognizes G as a nucleotide. A DNA-binding moduleincludes, for instance, a polypeptide which has sequence identity of85%, 90%, 95%, or 97% with the amino acid sequence of from position 1 toposition 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, or 32 and which substantially retains the function torecognize a single nucleotide.

In the first aspect of the polypeptide of the present invention, acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4n−3 countedfrom the N-terminus is the same for any of n. A combination of an aminoacid residue at position x and an amino acid residue at position y in aDNA-binding module at position 4n−2 counted from the N-terminus is thesame for any of n. A combination of an amino acid residue at position xand an amino acid residue at position y in a DNA-binding module atposition 4n−1 counted from the N-terminus is the same for any of n. Acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4n countedfrom the N-terminus is the same for any of n. In this context, n isnatural number of 1 to 10, preferably natural number of 1 to 7, morepreferably natural number of 1 to 5 and is preferably natural numberthat is sufficient for referring to all the DNA-binding modulescontained in a DNA-binding domain. In this context, x is natural numberof 1 to 40, preferably natural number of 1 to 10, more preferablynatural number of 2 to 6, even more preferably natural number of 3 to 5,most preferably natural number of 4. In this context, y is naturalnumber of 1 to 40, preferably natural number of 25 to 40, morepreferably natural number of 30 to 36, even more preferably naturalnumber of 31 to 33, most preferably natural number of 32. In thiscontext, x and y are different natural number from each other. Thevalues of x and y may vary depending on the length of a DNA-bindingmodule used. In this context, x preferably represents the numberindicating the position corresponding to the amino acid residue atposition 4 in a DNA-binding module consisting of 34 amino acid residueswhereas y preferably represents the number indicating the positioncorresponding to the amino acid residue at position 32 in a DNA-bindingmodule consisting of 34 amino acid residues.

In the first aspect of the polypeptide of the present invention, acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4n−3 countedfrom the N-terminus, a combination of an amino acid residue at positionx and an amino acid residue at position y in a DNA-binding module atposition 4n−2 counted from the N-terminus, a combination of an aminoacid residue at position x and an amino acid residue at position y in aDNA-binding module at position 4n−1 counted from the N-terminus, and acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4n countedfrom the N-terminus are different from each other. In this context, n isnatural number of 1 to 10, preferably natural number of 1 to 7, morepreferably natural number of 1 to 5 and is preferably natural numberthat is sufficient for referring to all the DNA-binding modulescontained in a DNA-binding domain. In this context, x is natural numberof 1 to 40, preferably natural number of 1 to 10, more preferablynatural number of 2 to 6, even more preferably natural number of 3 to 5,most preferably natural number of 4. In this context, y is naturalnumber of 1 to 40, preferably natural number of 25 to 40, morepreferably natural number of 30 to 36, even more preferably naturalnumber of 31 to 33, most preferably natural number of 32. In thiscontext, x and y are different natural number from each other.Preferably, a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position4n−3 counted from the N-terminus, a combination of an amino acid residueat position x and an amino acid residue at position y in a DNA-bindingmodule at position 4n−2 counted from the N-terminus, and a combinationof an amino acid residue at position x and an amino acid residue atposition y in a DNA-binding module at position 4n counted from theN-terminus are selected from the group consisting of a combination of Dand D, a combination of E and A, a combination of D and A, and acombination of A and D, respectively for x and y in this order.

In the second aspect, the present invention provides the polypeptide ofthe first aspect wherein the function domain is a DNA-cleaving domain.

In the third aspect, the present invention provides a vector comprisinga polynucleotide coding for the polypeptide of the first aspect or thesecond aspect. A vector includes a plasmid vector, a cosmid vector, aviral vector, an artificial chromosome vector and the like. Anartificial chromosome vector includes a yeast artificial chromosomevector (YAC), a bacterial artificial chromosome vector (BAC), a P1artificial chromosome vector (PAC), a mouse artificial chromosome vector(MAC), and a human artificial chromosome vector (HAC). A component of avector includes a nucleic acid such as DNA, RNA and the like, a nucleicacid analogue such as GNA, LNA, BNA, PNA, TNA and the like. A vector maybe modified with a component other than a nucleic acid such as asaccharide.

By introducing the vector of the third aspect of the present inventioninto cells and the like for expression, the polypeptide of the firstaspect or the second aspect of the present invention can be prepared.Also, by introducing the vector of the third aspect of the presentinvention into cells and the like for expression, a desired functioncorresponding to a function domain can be fulfilled in cells such as DNAmodification such as DNA recombination, DNA cleavage, etc.; expressionof other enzymatic activity such as transcriptional regulation;labelling of a DNA region by a reporter protein. In case that a functiondomain is a DNA-cleaving domain, by introducing plural, preferably two,of the vectors of the third aspect of the present invention into cellsand the like for expression, nucleotide sequence-specific double strandcleavage can be induced on a genomic DNA of the cells where the vectoris introduced so that mutation is introduced in the genome of the cells.The source of cells to which the vector of the third aspect of thepresent invention is introduced includes an animal such as mammal, e.g.Drosophila, zebrafish and mouse, a plant such as Arabidopsis thaliana,culture cells such as ES cells, iPS cells, and the like.

In the fourth aspect, the present invention provides a vector libraryfor preparing the vector of the third aspect. The vector library of thefourth aspect of the present invention is composed of a plurality ofvectors. The vector library preferably comprises vectors useful forpreparing the vector of the third aspect exhaustively with regard to acombination of four kinds of nucleotide which a combination of fourDNA-binding modules recognizes. However, as far as the manufacture ofthe vector of the third aspect is possible, the vector library maycomprise vectors not exhaustively. The vector library of the fourthaspect of the present invention comprises vectors for exhaustivelyconstructing the polypeptide of the first aspect or the second aspect ofthe present invention comprising e.g. 6 to 9, 10 to 13, 14 to 17, or 18to 21 DNA-binding modules. The polypeptide of the first aspect or thesecond aspect of the present invention, when having 14 to 21 DNA-bindingmodules, is particularly excellent in compatibility between a high levelof specificity of sequence recognition and a high level of function of afunction domain. Thus, such a vector library, though comprising a rathersmall number of vectors, is excellent as allowing for the manufacture ofthe polypeptide of the first aspect or the second aspect with higheffects by means of simple procedures.

All the vectors constituting the vector library of the fourth aspect ofthe present invention comprise a first restriction site, apolynucleotide coding for four DNA-binding modules and a secondrestriction site in this order from the 5′-end.

A combination of a first restriction site and a second restriction sitecontained in a vector constituting the vector library of the fourthaspect of the present invention is any one of a combination of arestriction site of type A and a restriction site of type B, acombination of a restriction site of type A and a restriction site oftype C, a combination of a restriction site of type A and a restrictionsite of type D, a combination of a restriction site of type A and arestriction site of type E, a combination of a restriction site of typeB and a restriction site of type C, a combination of a restriction siteof type C and a restriction site of type D, and a combination of arestriction site of type D and a restriction site of type E. Types A toE indicated in relation to a restriction site are used herein fordescriptive purposes for showing difference in property of therespective restriction sites. In case that the types are different fromeach other, property of their restriction sites is different whereas incase that the types are the same, property of their restriction sites isthe same. In the vector library of the fourth aspect of the presentinvention, the restriction sites of type A to type E are cleaved by thesame restriction enzyme. Also, the restriction sites of type A to type Eare cleaved by the same restriction enzyme to thereby produce cleavedterminals different from each other. Such a restriction site includesthe one by a restriction enzyme that cleaves an arbitrary site adjacentto a recognition site of the restriction enzyme, for instance, BsaI,Bbsl, BsmBI and the like.

As shown in FIG. 1, STEP 2, for the manufacture of the vector of thethird aspect of the present invention comprising 18 to 21 DNA-bindingmodules, the vector library of the fourth aspect of the presentinvention comprises a vector wherein a combination of a firstrestriction site and a second restriction site is a combination of arestriction site of type A and a restriction site of type B, a vectorwherein a combination of a first restriction site and a secondrestriction site is a combination of a restriction site of type B and arestriction site of type C, a vector wherein a combination of a firstrestriction site and a second restriction site is a combination of arestriction site of type C and a restriction site of type D, and avector wherein a combination of a first restriction site and a secondrestriction site is a combination of a restriction site of type D and arestriction site of type E, preferably exhaustively with regard tonucleotides recognized by DNA-binding modules.

Also as shown in FIG. 1, STEP 2, for the manufacture of the vector ofthe third aspect of the present invention comprising 14 to 17DNA-binding modules, the vector library of the fourth aspect of thepresent invention comprises a vector wherein a combination of a firstrestriction site and a second restriction site is a combination of arestriction site of type A and a restriction site of type C, a vectorwherein a combination of a first restriction site and a secondrestriction site is a combination of a restriction site of type C and arestriction site of type D, and a vector wherein a combination of afirst restriction site and a second restriction site is a combination ofa restriction site of type D and a restriction site of type E,preferably exhaustively with regard to nucleotides recognized byDNA-binding modules.

Besides, as shown in FIG. 1, STEP 2, for the manufacture of the vectorof the third aspect of the present invention comprising 10 to 13DNA-binding modules, the vector library of the fourth aspect of thepresent invention comprises a vector wherein a combination of a firstrestriction site and a second restriction site is a combination of arestriction site of type A and a restriction site of type D, and avector wherein a combination of a first restriction site and a secondrestriction site is a combination of a restriction site of type D and arestriction site of type E, preferably exhaustively with regard tonucleotides recognized by DNA-binding modules.

Furthermore, as shown in FIG. 1, STEP 2, for the manufacture of thevector of the third aspect of the present invention comprising 6 to 9DNA-binding modules, the vector library of the fourth aspect of thepresent invention comprises a vector wherein a combination of a firstrestriction site and a second restriction site is a combination of arestriction site of type A and a restriction site of type E, preferablyexhaustively with regard to nucleotides recognized by DNA-bindingmodules.

The vectors constituting the vector library of the fourth aspect of thepresent invention comprises DNA-binding modules. The length of aDNA-binding module includes, for instance, 30 to 38, 32 to 36, or 34.The length of DNA-binding modules contained in a DNA-binding domain ispreferably the same for all the vectors constituting the vector library.Also, the length of DNA-binding modules contained in a DNA-bindingdomain is preferably the same for all the four DNA-binding modulescontained in the vector constituting the vector library. A DNA-bindingmodule includes, for instance, a polypeptide consisting of the aminoacid sequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. A DNA-bindingmodule includes, for instance, a polypeptide which has sequence identityof 85%, 90%, 95%, or 97% with the amino acid sequence of from position 1to position 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, or 32 and which substantially retains the function torecognize a single nucleotide.

Among the four DNA-binding modules contained in vectors constituting thevector library of the fourth aspect of the present invention, acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 1 counted fromthe 5′-terminus is the same for an arbitrary vector constituting thevector library. Likewise, a combination of an amino acid residue atposition x and an amino acid residue at position y in a DNA-bindingmodule at position 2 counted from the 5′-terminus is the same for anarbitrary vector constituting the vector library. Also, a combination ofan amino acid residue at position x and an amino acid residue atposition y in a DNA-binding module at position 3 counted from the5′-terminus is the same for an arbitrary vector constituting the vectorlibrary. Also, a combination of an amino acid residue at position x andan amino acid residue at position y in a DNA-binding module at position4 counted from the 5′-terminus is the same for an arbitrary vectorconstituting the vector library. In this context, x is natural number of1 to 40, preferably natural number of 1 to 10, more preferably naturalnumber of 2 to 6, even more preferably natural number of 3 to 5, mostpreferably natural number of 4. In this context, y is natural number of1 to 40, preferably natural number of 25 to 40, more preferably naturalnumber of 30 to 36, even more preferably natural number of 31 to 33,most preferably natural number of 32. In this context, x and y aredifferent natural number from each other. The values of x and y may varydepending on the length of a DNA-binding module used. In this context, xpreferably represents the number indicating the position correspondingto the amino acid residue at position 4 in a DNA-binding moduleconsisting of 34 amino acid residues whereas y preferably represents thenumber indicating the position corresponding to the amino acid residueat position 32 in a DNA-binding module consisting of 34 amino acidresidues.

Among the four DNA-binding modules contained in vectors constituting thevector library of the fourth aspect of the present invention, acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 1 counted fromthe 5′-terminus, a combination of an amino acid residue at position xand an amino acid residue at position y in a DNA-binding module atposition 2 counted from the 5′-terminus, a combination of an amino acidresidue at position x and an amino acid residue at position y in aDNA-binding module at position 3 counted from the 5′-terminus, and acombination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module at position 4 counted fromthe 5′-terminus, are different from each other. In this context, x isnatural number of 1 to 40, preferably natural number of 1 to 10, morepreferably natural number of 2 to 6, even more preferably natural numberof 3 to 5, most preferably natural number of 4. In this context, y isnatural number of 1 to 40, preferably natural number of 25 to 40, morepreferably natural number of 30 to 36, even more preferably naturalnumber of 31 to 33, most preferably natural number of 32. In thiscontext, x and y are different natural number from each other. Thevalues of x and y may be for instance those as described above.Preferably, a combination of an amino acid residue at position x and anamino acid residue at position y in a DNA-binding module at position 1counted from the 5′-terminus is D and D, a combination of an amino acidresidue at position x and an amino acid residue at position y in aDNA-binding module at position 2 counted from the 5′-terminus is E andA, a combination of an amino acid residue at position x and an aminoacid residue at position y in a DNA-binding module at position 3 countedfrom the 5′-terminus is D and A, and a combination of an amino acidresidue at position x and an amino acid residue at position y in aDNA-binding module at position 4 counted from the 5′-terminus is A andD, respectively for x and y in this order.

By using the vector library of the fourth aspect of the presentinvention, the vector of the third aspect of the present invention canbe prepared with simple procedures. Specifically, vectors correspondingto the sequence of DNA-binding modules contained in the vector of thethird aspect of the present invention are selected from the vectorlibrary of the fourth aspect of the present invention, the selectedvectors are digested with restriction enzymes that cleave restrictionsites of types A to E and the vector fragments obtained by digestion arelinked together to prepare the vector of the third aspect of the presentinvention. All the vectors constituting the vector library of the fourthaspect of the present invention have two restriction sites, which arecleaved by the same restriction enzyme and produce cleaved terminalsdifferent from each other as a consequence of cleavage by said enzyme.Thus, for the manufacture of the vector of the third aspect of thepresent invention, digestion of the selected vectors and ligation of thevector fragments can be performed in one and the same reaction solution,respectively. Therefore, by using the vector library of the fourthaspect of the present invention, the vector of the third aspect of thepresent invention can be prepared with quite simple procedures.

In the fifth aspect, the present invention provides a vector set forpreparing the vector library of the fourth aspect.

The vector set of the fifth aspect of the present invention comprises aplurality of vectors. The vector set preferably comprises vectors usefulfor preparing the vector library of the fourth aspect exhaustively.However, as far as the manufacture of the vector library of the fourthaspect is possible, the vector set may comprise vectors notexhaustively.

All the vectors contained in the vector set of the fifth aspect of thepresent invention comprises a first restriction site, a DNA-bindingmodule and a second restriction site in this order from the 5′-end. Thefirst restriction site and the second restriction site are preferablythe ones not cleaved by a restriction enzyme that cleaves the firstrestriction site and the second restriction site contained in thevectors constituting the vector library of the fourth aspect of thepresent invention. In this regard, the vector of the third aspect can beprepared from the vector library of the fourth aspect with simplerprocedures.

The length of a DNA-binding module in the vector contained in the vectorset of the fifth aspect of the present invention is preferably the samefor all the vectors contained in the vector set. A DNA-binding moduleincludes, for instance, a polypeptide consisting of the amino acidsequence of from position 1 to position 34 of SEQ ID NOs:2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32. A DNA-binding moduleincludes, for instance, a polypeptide which has sequence identity of85%, 90%, 95%, or 97% with the amino acid sequence of from position 1 toposition 34 of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, or 32 and which substantially retains the function torecognize a single nucleotide.

The first restriction site and the second restriction site in the vectorcontained in the vector set of the fifth aspect of the present inventionare cleaved by the same restriction enzyme. The first restriction siteand the second restriction site are also cleaved by the same restrictionenzyme to thereby produce cleaved terminals different from each other.Such a restriction site includes the one by a restriction enzyme thatcleaves an arbitrary site adjacent to a recognition site of therestriction enzyme, for instance, BsaI, Bbsl, BsmBI and the like.

A combination of an amino acid residue at position x and an amino acidresidue at position y in a DNA-binding module in the vector contained inthe vector set of the fifth aspect of the present invention is any oneof four different combinations. The four different combinations of anamino acid residue at position x and an amino acid residue at position yincludes, for instance, a combination of D and D, a combination of E andA, a combination of D and A, and a combination of A and D, for x and yin this order. In this context, x is natural number of 1 to 40,preferably natural number of 1 to 10, more preferably natural number of2 to 6, even more preferably natural number of 3 to 5, most preferablynatural number of 4. In this context, y is natural number of 1 to 40,preferably natural number of 25 to 40, more preferably natural number of30 to 36, even more preferably natural number of 31 to 33, mostpreferably natural number of 32. In this context, x and y are differentnatural number from each other. The values of x and y may vary dependingon the length of a DNA-binding module used. In this context, xpreferably represents the number indicating the position correspondingto the amino acid residue at position 4 in a DNA-binding moduleconsisting of 34 amino acid residues whereas y preferably represents thenumber indicating the position corresponding to the amino acid residueat position 32 in a DNA-binding module consisting of 34 amino acidresidues.

The vectors contained in the vector set of the fifth aspect of thepresent invention include a vector in which a combination of the firstrestriction site and the second restriction site is a combination of atype a restriction site and a type β restriction site and a DNA-bindingmodule recognizes nucleotide of A, T, G, or C; a vector in which acombination of the first restriction site and the second restrictionsite is a combination of a type β restriction site and a type yrestriction site and a DNA-binding module recognizes nucleotide of A, T,G, or C; a vector in which a combination of the first restriction siteand the second restriction site is a combination of a type γ restrictionsite and a type δ restriction site and a DNA-binding module recognizesnucleotide of A, T, G, or C; a vector in which a combination of thefirst restriction site and the second restriction site is a combinationof a type δ restriction site and a type ε restriction site and aDNA-binding module recognizes nucleotide of A, T, G, or C. In thiscontext, types α to δ of the restriction site as used herein areexpediently set for showing difference in property of the restrictionsite, denoting that different types of the restriction site aredifferent in their property from each other whereas the same types ofthe restriction site are common in their property. The vector set of thefifth aspect of the present invention preferably include all the vectorsmentioned above. In this case, every mode of the vector library of thefourth aspect of the present invention can be prepared.

By using the vector set of the fifth aspect of the present invention,the vector library of the fourth aspect of the present invention can beprepared with simple procedures. Specifically, four vectors are selectedfrom the vector set of the fifth aspect of the present invention basedon a combination of the four DNA-binding modules contained in vectorsconstituting the vector library of the fourth aspect of the presentinvention, the selected vectors are digested with a restriction enzymethat cleaves the first restriction site and the second restriction siteand the vector fragments obtained by digestion are linked together toprepare the vector library of the fourth aspect of the presentinvention. All the vectors contained in the vector set of the fifthaspect of the present invention have two restriction sites, which arecleaved by the same restriction enzyme and produce cleaved terminalsdifferent from each other as a consequence of cleavage by said enzyme.Thus, for the manufacture of the vector library of the fourth aspect ofthe present invention, digestion of the selected vectors and ligation ofthe vector fragments can be performed in one and the same reactionsolution, respectively. Therefore, by using the vector set of the fifthaspect of the present invention, the vector library of the fourth aspectof the present invention can be prepared with quite simple procedures.

EXAMPLES

The present invention is further explained in more detail by means ofthe following Examples but is not limited thereto.

Example 1: Preparation of Vector Set

The nucleotide sequence shown in FIG. 2 with addition at both ends ofthe recognition site of restriction enzyme BsaI was prepared byartificial gene synthesis and inserted into pBluescript SK vector toprepare a vector set (p1HD-p4HD, p1NG-p4NG, p1NI-p4NI, p1NN-p4NN) foruse in STEP 1 of FIG. 1.

Example 2: Preparation of Vector Library

Using pFUS_B6 vector (Addgene) as a template, pFUS2 vector shown in STEP1 of FIG. 1 was prepared by In-Fusion cloning (Clontech). As shown inSTEP 1 of FIG. 1, using the prepared pFUS2 vector and the vector setprepared in Example 1, the Golden Gate reaction was performed to preparea vector library.

Example 3: Preparation of TALEN Expression Vector

Using pTALEN_v2 and pcDNA-TAL-NC2 (both Addgene), In-Fusion cloning wasperformed, a BsmBI site adjacent sequence for incorporating modules wasprepared by In-Fusion cloning, and a globin leader sequence wasintroduced upstream the initiation codon by In-Fusion cloning to prepareptCMV vectors as shown in FIG. 1, STEP 2. Using the prepared ptCMVvectors, the vector library prepared in Example 2, and the vectorscontained in Golden Gate TALEN and TAL Effector Kit, Yamamoto Lab TALENAccessory Pack (both Addgene), DNA-binding domains were inserted byGolden Gate procedure as shown in FIG. 1, STEP 2 to prepare TALENexpression vectors. The ptCMV vector was used in which the number ofamino acid residues of a region adjacent to the N-terminus ofDNA-binding domain (TALEN-N′) is 153 and the number of amino acidresidues of a region franked by the C-terminus of DNA-binding domain anda DNA-cleaving domain (TALEN-C′) is 47. FIG. 3 shows the nucleotide andamino acid sequences of an example of TALEN prepared.

As shown in FIG. 3, the amino acid residues at position 4 and atposition 32 of DNA-binding modules (34 amino acid residues in total) inthe TALEN expression vector of Example 3 were different from each otheramong DNA-binding modules at position 4n−3, at position 4n−2, atposition 4n−1 and at position 4n (n is natural number). The amino acidresidues at position 4 and at position 32 in DNA-binding modules atposition 4n−3 (n is natural number) were common among the respectiveDNA-binding modules. The same was applied to DNA-binding modules atposition 4n−3, at position 4n−2, at position 4n−1 and at position 4n (nis natural number). As such, by using the vector set of Example 1 andthe vector library of Example 2, the TALEN expression vectors could beprepared with a repetitive fashion for every four DNA-binding modules.

Comparative Example 1: Preparation of TALEN Expression Vector

The Golden Gate reaction was performed as described in Example 2 exceptthat pHD1-6, pNG1-6, pNI1-6, pNN1-6 contained in Golden Gate TALEN andTAL Effector Kit (Addgene) was used in place of the vector set preparedin Example 1 and that Yamamoto Lab TALEN Accessory Pack (Addgene) wasused as pFUS vector for use in the reaction, to prepare a vectorlibrary. Using the prepared vector library, the Golden Gate reaction wasperformed as described in Example 3 to prepare the TALEN expressionvectors.

Comparative Example 2: Preparation of TALEN Expression Vector

The procedures of Example 3 were carried out except that the ptCMVvector was used in which the number of amino acid residues of a regionadjacent to the N-terminus of DNA-binding domain (TALEN-N′) is 136 andthe number of amino acid residues of a region franked by the C-terminusof DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is 63, toprepare TALEN expression vectors.

Comparative Example 3: Preparation of TALEN Expression Vector

The procedures of Comparative Example 1 were carried out except that theptCMV vector was used in which the number of amino acid residues of aregion adjacent to the N-terminus of DNA-binding domain (TALEN-N′) is136 and the number of amino acid residues of a region franked by theC-terminus of DNA-binding domain and a DNA-cleaving domain (TALEN-C′) is63, to prepare TALEN expression vectors.

Test Example 1: Assessment of Recognition Specificity of TALEN

The TALEN expression vectors (L14 to L20 and R14 to R20) recognizing thesites indicated in FIG. 4B were prepared as in Example 3 or inComparative Examples 1 to 3. Each one from L14 to L20 and from R14 toR20 as prepared were combined together as shown in FIG. 4C to give rightand left TALEN expression vectors. Using the sequences shown in FIG. 4Bas a target sequence for TALEN, Single Strand Annealing Assay (cf.Non-patent reference 5) was conducted with HEK293T cell to assess theTALEN activity.

Specifically, Single Strand Annealing Assay was performed as describedbelow. First, a reporter vector was prepared in which a target sequenceof TALEN of interest was inserted into a reporter vector (pGL4-SSA;Addgene) wherein a segmented firefly luciferase gene was linkeddownstream CMV promoter. The target sequence of TALEN was prepared byannealing synthetic oligonucleotides and was inserted into pGL4-SSAvector treated with BsaI using Ligation-Convenience Kit (NIPPON GENECO., LTD.). Then, the prepared reporter vector together with the TALENexpression vector and pRL-CMV vector (Promega), which is an expressionvector of Renilla luciferase, were introduced into HEK293T cells on96-well plate by lipofectin procedure. After culture for 24 hours, thereporter activity was measured using Dual-Glo Luciferase Assay System(Promega). An amount of DNA introduced is each 200 ng for the right andleft TALEN expression vectors, 100 ng for the reporter vector, and 20 ngfor the pRL-CMV vector. Measurement of chemoluminescence was done withTriStar LB 941 plate reader (Berthold Japan K.K.).

FIG. 4C shows relative values of the reporter activity for therespective combinations of the right and left TALEN expression vectorsin Example 3 and Comparative Examples 1 to 3 in comparison with acombination of L20 and R17 in Comparative Example 1. Table of FIG. 4Bshows the length (the number of nucleotides) of the spacer regionfranked by the right and left TALEN recognition sites for the respectivecombinations of the right and left TALEN expression vectors.

As shown in FIG. 4C, in case of Example 3 and Comparative Example 1, aspecifically higher activity was obtained only for the limited cases of12 to 15 of the length of the spacer region, demonstrating that specificcleavage is possible for the limited spacer region. On the other hand,in case of Comparative Example 2 and Comparative Example 3, the level ofthe activity has no relevance with the length of the spacer region,demonstrating that the possibility is high that sequences with differentlength of the spacer region are recognized and cleaved. It was thusdemonstrated that, by using the TALEN expression vectors of Example 3,efficient DNA cleavage can be conducted with less non-specific cleavageof sequences with different length of the spacer region.

Test Example 2: Assessment of Activity of TALEN

A pair of the right and left TALEN expression vectors that recognize thesite shown in FIG. 5A (ATM (L17 for the left, R17 for the right), APC(L17 for the left, R17 for the right) and eGFP (L20 for the left, R18for the right)) were prepared as in Example 3 or Comparative Examples 1to 3. Using a pair of the right and left prepared by the respectiveprocedures as the right and left TALEN expression vectors, Single StrandAnnealing Assay (Non-patent reference 5) was performed with HEK293Tcells as in Test Example 1 using the sequence shown in FIG. 5A as atargeting sequence of TALEN to assess the activity of TALEN.

The results are shown in FIG. 5B in which the axis of ordinate indicatesrelative values of the reporter activity in comparison with acombination of L20 and R17 of Comparative Example 1 prepared in TestExample 1. As shown in FIG. 5B, in case of Example 3, a higher DNAcleavage activity of TALEN was observed for any of ATM, APC and eGPF ascompared to Comparative Example 1. This proved that the repetitivestructure of the DNA-binding modules in accordance with the presentinvention renders the DNA cleavage activity of TALEN be improved.

Test Example 3: Assessment of Recognition Specificity of TALEN

A pair of the right and left TALEN expression vectors that recognize thesite shown in FIG. 6A (L19 for the left, R18 for the right) wereprepared as in Example 3 or Comparative Examples 1 to 3. Using a pair ofthe right and left prepared by the respective procedures as the rightand left TALEN expression vectors, Single Strand Annealing Assay(Non-patent reference 5) was performed with HEK293T cells as in TestExample 1 using the sequence shown in FIG. 6A (no mismatches, 1 leftmismatch and 0 right mismatch (L:1 mismatch/R:0 mismatch), 1 leftmismatch and 1 right mismatch (L:1 mismatch/R:1 mismatch), or 2 leftmismatches and 2 right mismatches (L:2 mismatches/R:2 mismatches)) as atargeting sequence of TALEN to assess the activity of TALEN. In casethat the respective sequences shown in FIG. 6A are used as a targetingsequence of TALEN, mismatch occurs at lower cases in FIG. 6A and thusthe level of recognition specificity of TALEN used can be compared bycomparing the results of TALEN activity assessment of the targetingsequence of TALEN used.

The results are shown in FIG. 6B in which the axis of ordinate indicatesrelative values of the measurement of the firefly luciferase activitydivided by the measurement of the Renilla luciferase activity. As shownin FIG. 6B, in case that the TALEN expression vectors of ComparativeExample 2 were used, even in case of the sequence of 2 left mismatchesand 2 right mismatches, a high activity was observed and thus therecognition specificity of the TALEN expression vectors of ComparativeExample 2 was low. On the other hand, in case that the TALEN expressionvectors of Example 3 were used, in case of the sequence of 2 leftmismatches and 2 right mismatches, almost complete loss of the activitywas observed. This proved that the TALEN expression vectors of Example 3can afford to DNA cleavage with high specificity while maintaining ahigh cleavage activity as shown in Comparative Example 2. Therefore, itwas found that a target DNA of interest can be cleaved safely with highprobability by using the TALEN expression vectors of Example 3.

INDUSTRIAL APPLICABILITY

The present invention is useful e.g. for production of a variety ofsubstance by genetic engineering technique and can widely be used in thefield of medicine, engineering and agriculture.

1-2. (canceled)
 3. A vector library consisting of a plurality ofvectors, each of which vector has a first restriction site, apolynucleotide coding for four DNA-binding modules and a secondrestriction site in this order from the 5′-end, wherein a combination ofthe first restriction site and the second restriction site is any one ofa combination of a restriction site of type A and a restriction site oftype B, a combination of a restriction site of type A and a restrictionsite of type C, a combination of a restriction site of type A and arestriction site of type D, a combination of a restriction site of typeA and a restriction site of type E, a combination of a restriction siteof type B and a restriction site of type C, a combination of arestriction site of type C and a restriction site of type D, and acombination of a restriction site of type D and a restriction site oftype E, wherein each of the restriction sites of types A to E producedifferent cleaved terminals when cleaved with the same restrictionenzyme; and among the four DNA-binding modules, wherein a combination ofan amino acid residue at position 4 and an amino acid residue atposition 32 in a DNA-binding module at position 1 counted from the5′-terminus is the same for any of the vectors, wherein a combination ofan amino acid residue at position 4 and an amino acid residue atposition 32 in a DNA-binding module at position 2 counted from the5′-terminus is the same for any of the vectors, wherein a combination ofan amino acid residue at position 4 and an amino acid residue atposition 32 in a DNA-binding module at position 3 counted from the5′-terminus is the same for any of the vectors, wherein a combination ofan amino acid residue at position 4 and an amino acid residue atposition 32 in a DNA-binding module at position 4 counted from the5′-terminus is the same for any of the vectors, and wherein acombination of an amino acid residue at position 4 and an amino acidresidue at position 32 in a DNA-binding module at position 1 countedfrom the 5′-terminus, a combination of an amino acid residue at position4 and an amino acid residue at position 32 in a DNA-binding module atposition 2 counted from the 5′-terminus, a combination of an amino acidresidue at position 4 and an amino acid residue at position 32 in aDNA-binding module at position 3 counted from the 5′-terminus, and acombination of an amino acid residue at position 4 and an amino acidresidue at position 32 in a DNA-binding module at position 4 countedfrom the 5′-terminus are different from each other.
 4. A vector set forpreparing the vector library as set forth in claim 3, wherein the vectorset comprises a plurality of vectors, each of which vector comprises afirst restriction site, a DNA-binding module and a second restrictionsite in this order from the 5′-end, wherein the first restriction siteand the second restriction site produce different cleaved terminals whencleaved with the same restriction enzyme, wherein the first restrictionsite and the second restriction site are the ones not cleaved by arestriction enzyme that cleaves the first restriction site and thesecond restriction site contained in the vectors constituting the vectorlibrary as set forth in claim 3, and wherein a combination of an aminoacid residue at position 4 and an amino acid residue at position 32 in aDNA-binding module is any of the four different combinations.
 5. Amethod for preparing a modified cell which comprises introducing avector from the vector library as set forth in claim 3 into a cellfollowed by expression.
 6. A method for preparing a cell with mutationon the genome being introduced which comprises introducing a vector fromthe vector library as set forth in claim 3 into a cell followed byexpression.
 7. A modified cell produced by the method as set forth inclaim
 5. 8. A cell with mutation on the genome being introduced producedby the method as set forth in claim
 6. 9. A cell with modification by avector from the vector library as set forth in claim
 3. 10. A cell withmutation on the genome by a vector from the vector library as set forthin claim
 3. 11. A plant or an animal comprising the cell as set forth inclaim
 7. 12. A plant or an animal comprising the modified cell as setforth in claim
 8. 13. A plant or an animal comprising the cell as setforth in claim
 9. 14. A plant or an animal comprising the cell as setforth in claim 10.